Chemical constituents of Hedyotis pinifolia wall. collected in Thua Thien Hue

This study reports the chemical constituents from the whole plants Hedyotis pinifolia Wall. Ex G. Don (now accepted as Oldenlandia pinifolia (Wall. Ex G. Don) Kuntze) collected in Thua Thien Hue province. Thirteen compounds were isolated by chromatography method.

DOI: 10.15625/2525-2518/56/4/9625

CHEMICAL CONSTITUENTS OF HEDYOTIS PINIFOLIA WALL

COLLECTED IN THUA THIEN HUE

Khieu Thi Tam 1, 3 , Nguyen Thi Hoang Anh 1, 2, * , Nguyen Van Tuan 1, 4 ,

Dao Duc Thien 2 , Tran Duc Quan 2 , Nguyen Thanh Tam 1, 2 , Nguyen Chi Bao 5 ,

Trinh Thi Thuy 1, 2 , Tran Van Sung 2

1

Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Ha Noi

2

Institute of Chemistry, VAST, 18 Hoang Quoc Viet, Cau Giay, Ha Noi

3

Thai Nguyen University of Sciences, Thai Nguyen University, Tan Thinh, Thai Nguyen

4 Asean College, Van Lam, Hung Yen 5

Department of Chemistry, Hue University, 77 Nguyen Hue, Hue City

*

Email: hoanganhvhh@gmail.com

Received: 1 February 2018; Accepted for publication: 10 May 2018

Abstract This study reports the chemical constituents from the whole plants Hedyotis pinifolia

Wall Ex G Don (now accepted as Oldenlandia pinifolia (Wall Ex G Don) Kuntze) collected

in Thua Thien Hue province Thirteen compounds were isolated by chromatography method

Their structures were elucidated using MS and NMR analysis and compared with reported data

They contain three anthraquinones, a carotenoid, two triterpenes, four iridoid glucosides and

three flavonoid glycosides Three of them were found for the first time in this genus

Keywords: Hedyotis pinifolia (Oldenlandia pinifolia), anthraquinone, carotenoid, triterpene,

iridoid glucosides and flavonoid glycosides

Classification numbers: 1.1.1, 1.1.6, 1.2.1

1 INTRODUCTION

The Hedyotis genus belongs to the Rubiaceae family, which has about 180 species and is

native to tropical and subtropical Asia [1] Numerous Hedyotis species are used in traditional

medicine for the treatment of inflammation, cancer and other diseases [2-4] such as Hedyotis

biflora, H corymbosa, H diffusa, H verticillata, etc For example, the leaves of H auricularia

were used for treatment of diarrhea, dysentery in India, while using as vegetable with reducing

blood pressure effect in Sri Lanka [5] Vietnamese folk medicine used H herbacea as

expectorant, hypothermia and tonic; H tenelliflora as detoxify, analgesic agents [4] From this

genus, many compounds with the novel structures and unique biological activities of the

alkaloid, anthraquinone, iridoid, triterpenoid and lignin classes have been reported [6-9]

Hedyotis pinifolia is a small herb growing in sandy areas from Hue to the south of Viet Nam

[10] Until now, according to to our literature search, there has been only one report on the

phytochemistry of this species [11] In this paper, we isolated the chemical constituents from the

n-hexane, ethyl acetate and n-butanol extracts of H pinifolia The structures of isolated

compounds have been elucidated as three anthraquinones: 2-hydroxy-1-methoxy-anthraquinone

(1); 1,6-dihydroxy-2-methylanthraquinone (2); digiferruginol (3); one carotenoid: lutein (4); two

triterpenes: ursolic acid (5), oleanolic acid (6); four iridoid glycosides: asperuloside (7),

deacetyl asperuloside (8), asperulosidic acid (9), scandoside methyl ester (10); three flavonoid

glycosides: afzelin (11), rutin (12), isorhamnetin-3-O-β-rutinoside (13) Among them,

compounds 1, 3 and 11 were isolated for the first time in this genus

2 MATERIALS AND METHODS

2.1 Equipments and methods

ESI-MS: LC-MSD-Trap-SL NMR: Bruker Avance 500 MHz (1H) and 125 MHz (13C) The

1

H chemical shifts were referenced to the internal TMS; the 13C chemical shifts to the solvent

signals The 2D experiments (HSQC and HMBC) were performed using standard Bruker pulse

sequences at room temperature Analytical TLC was performed on silica gel 60 F254 plates

(Merck) Spots were visualized using UV light and vanillin-H2SO4 reagent For preparative

column chromatography silica gel 60, 60-200 µm (Merck) and sephadex LH-20 were used

2.2 Plant material

Hedyotis pinifolia was collected on October 2014 in Phu Vang, Thua Thien Hue province

of Viet Nam and determined by Dr Do Xuan Cam, Hue University The voucher specimen

(VHH.TTH 10.2014.1) is deposited at the Institute of Chemistry, Vietnam Academy of Sciences

and Technology (VAST)

2.3 Extraction and isolation

The air-dried whole of H pinifolia (2.1 kg) were ground and extracted three times with

95 % MeOH at room temperature The organic solvent was evaporated under reduced pressure

and the aq solution was partitioned with n-hexane, ethyl acetate and n-butanol, successively

The n-hexane extract (36.2 g) was given on silica gel column, eluting with gradient n-hexane: EtOAc (from 100 % n-hexane to 100 % EtOAc) to yield 10 fractions (H1-H10)

Fraction H7 (150 mg) was chromatographed on silica gel column, n-hexane: EtOAc (15:1),

followed by Sephadex LH-20, CH2Cl2: MeOH (1:9) to furnish compound 1 (5 mg) Compound 2

(6 mg) and 4 (11 mg) were isolated when fraction H10 (410 mg) was purified over Sephadex

LH-20, using CH2Cl2: MeOH (1:9) as eluent

The ethyl acetate extract (34.2 g) was chromatographed over silica gel using gradient

n-hexane:EtOAc to yield 7 fractions (E1-E7) Fraction E2 (4.42 g) was repurified over silica gel

column, CH2Cl2: MeOH (10:1) to give 30 mg of 5 Rechromatography of fraction E3 (1.62 g)

was done on silica gel column, CH2Cl2: MeOH (10:1) furnished compound 6 (200 mg) Fraction

E4 (1.15 g) was given on silica gel column, eluting with CH2Cl2: MeOH (9.5:1), followed on

Sephadex LH-20 to obtain compound 3 (10 mg)

The n-butanol extract (32.0 g) was purified over silica gel columns to obtain 12 fractions

(B1-B12) The fraction B3 (2.9 g) was rechromatographed on Sephadex LH-20 column with solvent systems of CH2Cl2: MeOH (1:9) to yield three subfractions (B3.1 – B3.3) Compound 7

(30 mg) was isolated by purification of subfraction B3.2 using Sephadex LH-20 column, MeOH The purification of fraction B4 (1.35 g) on Sephadex LH-20 column, eluated with CH2Cl2:

MeOH (1:9) to give compound 11 (10 mg) The purification of fraction B5 (2.42 g) was carried

out on Sephadex LH-20 column with solvent systems of CH2Cl2:MeOH (1:9) to give two subfractions (B5.1-B5.2) Repeated chromatography of the subfraction B5.2 on sephadex LH-20

column, MeOH yielded compound 10 (16 mg) The fraction B6 (2.95 g) was given over

Sephadex LH-20 column eluated with MeOH to furnish compound 8 (11 mg) The fraction B9

(3.9 g) was chromatographed on Sephadex LH-20 column, eluated with solvent systems of

CH2Cl2: MeOH (1:9) to yield compound 12 (40 mg) The fraction 10 (1.5 g) was given on

Sephadex LH-20 column, eluated with CH2Cl2: MeOH (1:9) to furnish 2 subfractions (B10.1

and B10.2) Compound 7 (10 mg) and 13 (10 mg) were obtained by repeated chromatography of

subfraction B10.2 The fraction 12 (1.5 g) was chromatographed on Sephadex LH-20 column, eluated with CH2Cl2: MeOH (1:9) to give two subfractions (B12.1-B12.2) Rechromatography of

B12.2 on sephadex LH-20 column, MeOH furnished compound 9 (10 mg)

2-hydroxy-1-methoxy-anthraquinone (1): Orange-red needles (-)-ESI-MS m/z: 253 [M-H]

-1

H NMR (500 MHz, CDCl3): δH (ppm)7.36 (1H, d, 9.0 Hz, H-3), 8.14 (1H, d, 9.0 Hz, H-4), 8.27 (2H, m, H-5, H-8), 7.74 (2H, m, H-6, H-7), 4.04 (3H, s, OMe), 6.69 (1H, s, OH)

13

C NMR (125 MHz, CDCl3): δC (ppm)146.6 (C-1), 155.6 (C-2), 120.3 (C-3), 125.8 (C-4), 127.1 (C-5), 133.9 (C-6, C-7), 126.9 (C-8), 182.7 (C-9), 182.1 (C-10), 133.0 (C-11), 134.5 (C-12), 125.7 (C-13), 127.6 (C-14), 62.3 (OMe)

1,6-dihydroxy-2-methylanthraquinone (2): Orange powder (-)-ESI-MS m/z: 253 [M-H]

-1

H NMR (500 MHz, DMSO-d6): δH (ppm)7.61 (1H, d, 7.5 Hz, H-3), 7.55 (1H, d, 7.5 Hz, H-4), 7.44 (1H, d, 2.5 Hz, H-5), 7.21 (1H, dd, 2.5, 8.5 Hz, H-7), 8.08 (1H, d, 8.5 Hz, H-8), 13.08 (1H,

s, 1-OH), 2.27 (3H, s, 2-CH3)

13

C NMR (125 MHz, DMSO-d6): δC (ppm) 159.9 (C-1), 114.6 (C-2), 136.8 (C-3), 118.6 (C-4), 112.5 (C-5), 163.9 (C-6), 121.4 (C-7), 129.8 (C-8), 187.6 (C-9), 181.7 (C-10), 131.1 (C-4a), 124.4 (C-8a), 134.2 (C-9a), 135.6 (C-10a), 15.7 (2-CH3)

Digiferruginol (3): Orange-yellow needles (-)-ESI-MS m/z: 253 [M-H]-

1

H NMR (500 MHz, DMSO-d6): δH (ppm) 7.77 (1H, d, 7.5 Hz, H-3), 7.92 (1H, d, 8.0 Hz, H-4), 8.20 (1H, m, H-5), 7.95 (2H, m, H-6, H-7), 8.25 (1H, m, H-8), 4.66 (2H, s, CH2OH), 5.46 (1H, t,

5.5 Hz, CH2OH), 12.77 (1H, s, OH)

13

C NMR (125 MHz, DMSO-d6): δC (ppm) 158.4 (C-1), 138.2 (C-2), 131.3 (C-3), 118.8 (C-4), 126.8 (C-5), 134.5 (C-6), 135.1 (C-7), 126.6 (C-8), 188.7 (C-9), 181.8 (C-10), 133.6 (C-5a), 133.2 (C-8a), 114.9 (C-9a), 132.8 (C-10a), 57.4 (CH2OH)

Lutein (4): Orange-red powder (+)-ESI-MS m/z: 569 [M+H]+

1

H-, 13C-NMR (500, 125 MHz, CDCl3): Table 1

Ursolic acid (5): White amorphous powder (-)-ESI-MS m/z: 455 [M-H]

-1

H-NMR (500 MHz, CDCl3): δH (ppm) 2.98 (m, H-3), 5.11 (m, H-12), 2.09 (d, 11.3 Hz, H-18), 0.88 (s, Me-23), 0.66 (s, Me-24), 0.85 (s, Me-25), 0.73 (s, Me-26), 1.02 (s, Me-27), 0.79 (d, 6.4

Hz, Me-29), 0.89 (d, 8.7 Hz, Me-30)

C NMR (125 MHz, CDCl3): δC (ppm) 38.2 1), 27.0 2), 76.8 3), 38.4 4), 54.8 (C-5), 18.0 (C-6), 30.2 (C-7), 39.1 (C-8), 47.0 (C-9), 36.5 (C-10), 23.8 (C-11), 124.6 (C-12), 138.2 13), 41.6 14), 32.7 15), 22.8 16), 46.8 17), 52.4 18), 38.4 19), 38.5 (C-20), 27.5 (C-21), 36.3 (C-22), 28.3 (C-23); 16.9 (C-24); 16.1 (C-25), 15.2 (C-26), 23.3 (C-27),

178.3 (C-28), 17.0 (C-29), 21.1 (C-30)

Oleanolic acid (6): White amorphous powder (-)-ESI-MS m/z: 455 [M-H]

-1

H NMR (500 MHz, CDCl3): δH (ppm) 5.27 (1H, t, 3.5Hz, 12), 3.20 (1H, dd, 4.0, 11.0 Hz, H-3), 2.81 (1H, dd, 4.0, 13.5 Hz, H-18), 1.12, 0.97, 0.91, 0.90, 0.89, 0.76, 0.74 (each 3H, s, Me-23,

24, 25, 26, 27, 29, 30)

13

C NMR (125 MHz, CDCl3): δC (ppm) 38.4 1), 27.7 2), 79.1 3), 38.8 4), 55.3 (C-5), 18.3 (C-6), 32.7 (C-7), 39.3 (C-8), 47.7 (C-9), 37.1 (C-10), 23.0 (C-11), 122.7 (C-12), 143.6 13), 41.7 14), 27.2 15), 23.4 16), 46.5 17), 41.1 18), 45.9 19), 30.7 (C-20), 33.8 (C-21), 32.5 (C-22), 28.1 (C-23), 15.6 (C-24), 15.3 (C-25), 17.1 (C-26), 25.9 (C-27), 181.6 (C-28), 33.1 (C-29), 23.6 (C-30)

Asperuloside (7): white powder (+) ESI-MS: m/z = 437 [M+Na]+ NMR data: Table 2

Deacetyl asperuloside (8): white powder (-) ESI-MS: m/z = 371 [M-H]- NMR data: Table 2

Scandoside methyl ester (9): white powder (+) ESI-MS: m/z = 427 [M+Na]+ NMR data: Table

2

Asperulosidic acid (10): white powder (+) ESI-MS: m/z = 455 [M+Na]+ NMR data: Table 2

Afzelin (11): yellow powder (-) ESI-MS: m/z = 431 [M-H]-

1

H-NMR (500 MHz, CD3OD): δH (ppm) 6.22 (1H, d, 2.0 Hz, H-6), 6.40 (1H, d, 2.0 Hz, H-8), 7.79 (2H, d, 9.0 Hz, H-2’, 6’), 6.96 (2H, d, 9.0 Hz, H-3’, 5’), 5.40 (1H, d, 1.5 Hz, H-1”), 3.73 (1H, dd, 3.0, 9.0 Hz, H-2”); 3.36 (1H, d, 5.0 Hz, H-3”), 3.35 (1H, d, 5.0 Hz, H-4”), 4.24 (1H, dd, 2.0, 4.0 Hz, H-5”); 0.94 (3H, d, 6.0 Hz, H-6”)

13

C-NMR (125 MHz, CD3OD): δC (ppm) 159.3 (C-2), 136.2 (C-3), 179.6 (C-4); 163.2 (C-5), 99.9 (C-6), 166.1 (C-7), 94.8 (C-8), 158.6 (C-9), 105.9 (C-10), 122.7 (C-1’), 131.9 (C-2’, C-6’), 116.5 (C3’, C-5’), 161.6 (C-4’), 103.5 (C-1”), 72.0 (C-2”), 72.2 (C-3”), 73.2 (C-4”), 71.9 (C-5”), 17.6 (C-6”)

Rutin (12): yellow powder (-) ESI-MS: m/z = 609 [M-H]-

1

H-NMR (500 MHz, CD3OD): δH (ppm)6.22 (1H, br s, H-6), 6.42 (1H, br s, H-8), 7.71 (1H, br

s, H-2’), 6.91 (1H, d, 8.0 Hz, H-5’), 7.64 (1H, br d, 8.0 Hz, H-6’), 5.07 (1H, d, 7.5 Hz, H-1”), 3.53 (1H, t, 9.0 Hz, H-2”), 3.81 (1H, br d, 9.5 Hz, H-6”), 4.54 (1H, br s, H-1”’), 3.69 (1H, br s, H-2”’), 3.58 (1H, dd, 3.5, 9.5 Hz, H-3”’), 3.31 (1H, m, H-4”’), 3.44 (1H, m, H-5”’), 1.14 (3H, d,

6.5 Hz, H-6”’)

13

C-NMR (125 MHz, CD3OD): δC (ppm) 158.4 (C-2), 135.6 (C-3), 179.3 (C-4), 162.7 (C-5), 100.0 (C-6), 166.0 (C-7), 95.0 (C-8), 159.4 (C-9), 105.6 (C-10), 123.1 (C-1’), 117.7 (C-2’), 145.6 (C-3’), 149.7 (C-4’), 116.1 (C-5’), 123.6 (C-6’), 104.8 (C-1”), 75.5 (C-2”), 78.0 (C-3”), 71.3 (C-4”), 77.1 (C-5”), 68.6 CH2 6”), 102.3 1”’), 72.0 2”’); 72.1 3”’); 73.9 (C-4”’), 69.6 (C-5”’), 17.8 (C-6”’)

Isorhamnetin-3-O-β-rutinoside (13): yellow powder (-) ESI-MS: m/z = 623 [M-H]-

1

H-NMR (500 MHz, CD3OD): δH (ppm)6.22 (1H, d, 1.5 Hz, H-6), 6.41 (1H, d, 1.5 Hz, H-8), 7.95 (1H, d, 2.0 Hz, H-2’), 6.93 (1H, d, 8.5 Hz, H-5’), 7.64 (1H, dd, 2.0, 8.5 Hz, H-6’), 5.24

(1H, d, 7.5 Hz, H-1”), 4.55 (1H, d, 1.0 Hz, H-1”’), 1.12 (3H, d, 7.5 Hz, H-6”’), 3.96 (3H, s,

OMe)

13

C-NMR (125 MHz, CD3OD): δC (ppm) 158.5 (C-2), 135.5 (C-3), 179.3 (C-4), 162.9 (C-5), 100.0 (C-6), 166.0 (C-7), 94.9 (C-8), 158.9 (C-9), 105.7 (C-10), 123.0 (C-1’), 114.6 (C-2’), 148.3 (C-3’), 150.8 (C-4’), 116.1 (C-5’); 124.0 (C-6’), 104.4 (C-1”), 75.9 (C-2”), 78.1 (C-3”), 71.6 (C-4”), 77.3 (C-5”), 68.5 (C-6”), 102.49 (C-1”’), 72.0 (C-2”’), 72.3 (C-3”’), 73.8 (C-4”’), 69.8 (C-5”’), 17.9 (C-6”’), 56.8 (-OMe)

3 RESULTS AND DISCUSSION

Thirteen compounds were isolated from n-hexane, ethyl acetate and n-butanol extracts of the whole plant of H pinifolia by repeated column chromatography with the appropriate solvent

systems Their structures were identified as 2-hydroxy-1-methoxy-anthraquinone (1); 1,6-dihydroxy-2-methylanthraquinone (2); digiferruginol (3); lutein (4); ursolic acid (5) and oleanolic acid (6) asperuloside (7), deacetyl asperuloside (8), asperulosidic acid (9), scandoside

methyl ester (10), afzelin (11), rutin (12), isorhamnetin-3-O-β-rutinoside (13) by the analysis of

their 1D, 2D-NMR, ESI-MS spectra and compared with published data

The structure of isolated compounds (1 -13)

O

O OH

O

OH

OH

CH3 H3C

C

O

C

H3

C

OH

4

HMBC correlations of compounds 1, 3, 4

Compound 1 obtained as orange-red needles, gave pseudo molecular peak at m/z = 253

[M-H]- in the negative ESI-MS The 1H NMR spectrum indicated the characteristic signals of the

anthraquinone type, including the signals of the two aromatic rings: two ortho-coupled aromatic protons at δH 7.36 (d, J = 9.0 Hz, H-3); 8.14 (d, J = 9.0 Hz, H-4) of the first ring and typical

aromatic protons of the A2B2 substituted ring at δH 8.27 (2H, m, H-5, H-8); 7.74 (2H, m, H-6, H-7) of the second together with an aromatic methoxy group at δH 4.04 Beside these signals, the 13C NMR spectrum gave the signals of the two carbonyl carbons at δC 182.7 and 182.1 and

six aromatic quaternary carbons comprising one hydroxy-carbon at δC 155.6; a carbon connected

to a methoxy group at δC 144.6 and four others The position of the hydroxy group at C-2 was

deduced from HMBC correlations among signals at δH 6.69 (2-OH), δC 120.3 (C-3); 155.6 (C-2)

and 146.6 (C-1); among signals at δH 7.36 (H-3), δC 146.6 (C-1) and 127.6 (C-4a) The structure

of 1 was thus determined to be 2-hydroxy-1-methoxy-anthraquinone or alizarin-1-methyl ether

when compared to the literature [12] This compound was isolated previously from Hedyotis diffusa and inhibited protein tyrosine kinases v-src and pp60src and the growth of Bcap37

cell line (IC50 65 µM) Furthermore, it could induce apoptosis on SPC-1-A cell (IC50 79 µM) with a close relationship to the mitochondrial apoptotic pathway [13]

Compound 2 was isolated as an orange powder, showed [M-H]- peak at m/z = 235 in the

negative ESI-MS Its NMR spectra showed characteristic signals of an 9,10-anthraquinone very

similar to those of 1, revealing two carbonyl carbons at δC 187.6 and 181.7 as well as proton

signals of two aromatic rings: two ortho-coupled aromatic protons and typical aromatic protons

of the 1,3,4-substituted ring These signals suggested that the substituted positions of 2 are the same in compound 1 but with other groups The connection of hydroxy group at C-1 was

confirmed based on a chelated hydroxy proton in 1H NMR (measured in DMSO-d 6 ) at δH 13.08,

as well as the downshifted carbonyl carbon at δC 187.6 The position of the hydroxy group at C-6

was deduced from HMBC correlations among signals at δH 8.08 (H-8), δC 187.6 (C-9) and 163.9

(C-6); among signals at δH 7.44 (H-5), δC 181.7 (C-10) and 121.4 (C-7); and among signals at δH

7.21 (H-7), δC 112.5 (C-5) and 124.4 (C-8a) An aromatic methyl group [δH 2.27 (3H, s); δC

15.7] was determined to connect at C-2 by correlations among signals at δH 2.27 (CH3), δC 159.9

(C-1), 136.8 (C-3) The spectroscopic data of 2 was identical to those of

1,6-dihydroxy-2-methylanthraquinone in the literature [14] This compound was first isolated from Cinchona pubescens in 1986 [14]

Table 1 1H- and 13C-NMR (500 and 125 MHz) data of compound 4 and lutein (CDCl3)

No δH (ppm, J, Hz) δC (ppm) δH (ppm, J, Hz) δC (ppm)

Compound 4 Lutein [CDCl 3 ]

2 1.48 (t, 12.0) 48.5 1.48 (t, 12.0) 48.4

4 ax

4 eq

2.04 (dd, 17.0; 10.0)

2.33-2.42 (m)

42.6 2.04 (dd, 17.0; 10.0)

2.33-2.45 (m)

42.5

11 6.58-6.67 (m) 124.8 6.55-6.71 (m) 124.9

12 6.36 (d, 15.0) 137.7 6.36 (d, 15.0) 137.6

14 6.25 (br d, 9.0) 132.6 6.26 (m) 132.6

15 6.58-6.67 (m) 130.1 6.55-6.71 (m) 130.0

1-gem

Me

2’ ax

2’ eq

1.37 (dd, 13.0; 7.0)

1.84 (dd, 13.0; 6.0)

44.6 1.37 (dd, 13.0; 7.0)

1.84 (dd, 13.0; 6.0)

44.7

6’ 2.33 – 2.42 m 55.0 2.33-2.45 (m) 55.0 7’ 5.43 (dd, 10.0; 15.5) 128.7 5.43 (dd, 15.5; 10.0) 128.6

11’ 6.58-6.67 (m) 124.5 6.55-6.71 (m) 124.5 12’ 6.36 (d, 15.0) 137.6 6.36 (d, 15.0) 137.6

14’ 6.25 (br d, 9.0) 132.6 6.26 (m) 132.6 15’ 6.58-6.67 (m) 130.0 6.55-6.71 (m) 130.0

Compound 3 was obtained as yellow needle, showed [M-H]- peak at m/z = 235 in the

negative ESI-MS Its NMR spectra predicted to be an 9,10-anthraquinone from similar signals

to those of 1 Beside the same signals as in 1, the NMR spectra of 3 appeared additional

characteristic signals of the hydroxy group at δH 12.77 and hydroxymethylen group [δH 4.66 (d,

J = 5.0 Hz, 2 -CH2OH); 5.46 (t, J = 5.5 Hz, 2-CH2OH); δC 57.4 The position of the hydroxyl group at C-1 and hydroxymethylen group at C-2 was deduced from HMBC correlations

Consequently, 3 was identified as digiferruginol by comparison with the published data [15]

Digiferruginol exhibited a significant cytotoxic activity against KB cancer cell line with an ED value of 0.09 µg/mL [15]

Compound 4 was isolated as an amorphous red-orange powder, indicated [M+H]+ peak at

m/z = 569 in the positive ESI-MS The NMR spectrum of 4 appears characteristic signals of a

carotenoid including conjugated olefins at δH 5.43-6.67; δC 124.5-138.5 together with 4 methylene groups Besides, two hydroxy-methine groups [(δH 4.00 (1H, m, H-3); δC 65.1 (C-3)

and 4.25 (1H, m, H-3’); 65.9 (C-3’)] and 10 methyl groups were observed The connection of

two hydroxy groups at C-3 and C-3’ was confirmed based on the downshifted carbons with δC

65.1 (C-3) and 65.9 (C-3’) The position of the allene moiety was determined to connect to alicyclic moiety at C-6 by HMBC correlations among signals at δH 6.12 (H-7) to δC 126.2 (C-5) and 137.6 (C-6) The conjugated alkadiene chain was deduced from HMBC correlations among protons H-14 with C-12 and C-15’; H-14’ with C-12’, C-15 and methyl group carbon connected

to C-13’ The structure of 4 is concluded as lutein when compared with the spectral data in the

literature [16] Lutein plays significant roles in human health, particularly for eyes, linked to reduced risk of age-related macular degeneration and cataracts [17]

The structures of compounds 5 and 6 were elucidated as ursolic acid and oleanolic acid,

respectively, by comparison with authentic sample in thin layer chromatography and 1H, 13C NMR spectral data [18, 19] Oleanolic acid and ursolic acid are triterpenoid compounds that widely

shown pharmacological activities, such as hepatoprotective, anti-inflammatory, antioxidant, and anticancer effects [20]

Compound 7 showed the pseudo molecular peak at m/z = 437 [M+Na]+ in the positive

ESI-MS spectrum The molecular formula of C18H22O11 was deduced from MS and NMR data NMR spectra showed the signals of two double bonds -CH=C< at δH = 7.32 (1H, d, 2.0 Hz), δC = 150.3 CH, 106.1 C and δH = 5.75 (1H, br s), δC = 128.9 CH, 144.2 C; two oxygenated methine at

δH = 5.97 (1H, d, 1.0 Hz), δC = 93.3 and δH = 5.59 (1H, br d, 6.5 Hz), δC = 86.3 CH Besides these, two methine and an oxy-methylene have been observed in NMR spectra The 13C NMR spectrum indicated the signals of a lactone and an acetyl groups, revealing by signals at δC = 172.3 C and 172.6 C, 20.6 CH3, respectively The presence of one β-D-glucopyranose unit in the molecule was confirmed by typical signals of anomer group at δH = 4.71 (1H, d, 8.0 Hz), δC

= 100.0 and methylene group (Glc-6) at δH = 3.94 (dd, 12.0 & 2.0 Hz), 3.70 (dd, 12.0 & 6.0 Hz),

δC = 62.8 The correlations in the HMBC spectrum between H-1 (δH 5.97) with C-3, C-5, C-8, C-9 and Glc-1; H-3 (δH 7.32) with C-1, C-4, C-5 and C-11; H-7 (δH 5.75) with 5, 6, 8,

C-9 and C-10 determined the substituted positions in compound 7 The above analysis and compared with the published data [21] led to conclude that 7 is asperuloside, a glucoside of

iridoid C-9, which was already isolated from Galium verum [22]

Compound 8 was obtained as white powder The pseudo molecular ion peak at m/z = 371

[M-H]- in the negative ESI-MS and NMR spectra led to conclude that the molecular formula of 8

is C16H20O10 Its NMR data were very similar to those of 7, suggested it is an iridoid glucoside

too The only difference between two compounds revealed by the replacement of acetyl signal

group in 7 by hydroxy-methylene signals in compound 8 (δH = 4.21, 2H, br s, δC = 60.1) The loss of 42 mass units (CH3CO-) of compound 2 compared with 7 confirmed the above mentioned suggestion Therefore, the structure of 8 was determined as deacetyl asperuloside by

comparison with the data in literature [21]

Compound 9 was isolated as white powder The positive ESI-MS gave pseudo molecular

ion peak at m/z 427 [M+Na]+ Its molecular formula was determined as C17H24O11 from MS and

NMR data NMR spectra of 9 showed similar signals to those of 8 The upshifted of C-6 (δC

82.6) and C-11 (δC 170.3) together with the addition of methoxy group at δH = 3.77, δC = 52.0

suggested that lactone group in 8 was opened to form hydroxyl and methyl ester groups in 9

Thus, the structure of 9 was elucidated as scandoside methyl ester when compared with pulished

data in [21]

NMR spectra of 10 suggested that is an iridoid glucoside, very similar to those of 9 with

iridoid and β-D-glucopyranose signals But, the methyl ester and hydroxy signals in 9 were

replaced by carboxylic at δC 170.0 (C-11) and acetyl at 172.5, 2.03/20.8 The position of substituted groups were confirmed by HMBC correlations These analysis combined the

published data in [23] led to conclude that 10 is asperulosidic acid This compound exhibited

cytotoxic activity on HL-60 and HCT15 cancer cell lines [24]

Compound 11 was obtained as yellow powder The molecular formula was determined as

C21H20O10 from pseudo molecular ion peak at m/z = 431 [M-H]- in the negative ESI-MS and NMR spectra The 1H-NMR spectrum exhibited two meta-coupled protons at δH 6.22 (1H, d, J = 2.0 Hz, H-6), 6.40 (1H, d, J = 2.0 Hz, H-8), characteristic proton signals of para-substituted

aromatic ring at δH 7.79 (2H, d, J = 9.0 Hz, H-2’, 6’); 6.96 (2H, d, J = 9.0 Hz, H-3’, 5’) Besides

these, the presence of an α-L-rhamnopyranose have been observed, revealing by a proton anomer at δH 5.40 (1H, d, J = 1.5 Hz) and a methyl group at δH 0.94 (3H, d, J = 6.0 Hz) The

13

C-NMR spectrum of 11 indicated 21 carbons, among them 15 belong to flavonoid skeleton and

6 carbons of sugar unit The above analysis combined the comparison with the published data

[25] led to conclude that 11 is 3-O-α-L-rhamnopyranoside kaempferol, named afzelin It was

isolated for the first time from Afzelia doussie [26] Afzelin inhibited the growth of prostate cancer cells in vitro [27]

Compound 12 was isolated as yellow powder and showed the pseudo molecular ion peak at

m/z = 609 [M-H]- in the negative ESI-MS, corresponds to C27H30O16 Its NMR spectra indicated that is a flavonoid glycoside, revealing by the signals of two meta-protons in A ring at δH = 6.22

br s (H-6), 6.42 br s (H-8), three protons of ABX spin system in ring B at δH = 7.71 br s (H-2’), 6.91 d, 8.0 Hz (H-5’), 7.64 br d, 8.0 Hz (H-6’), ketone group in ring C at δC = 179.3 (C-4) Furthermore, the characteristic signals of β-D-glucopyranose unit at δH = 5.07 d, 7.5 Hz (H-1”),

δC = 104.8 (C-1”) and α-L-rhamnopyranose at δH = 4.54, br s (H-1”’), 1.14 d, 6.5 (H-6”’); δC =

102.3 (C-1”’), 17.8 (C-6”’) have been observed The NMR data of 12 are identical with those of

rutin in the literature [28] Rutin exists widely in the nature, for example in barley, fruit types of

Citrus genus and medicinal plant as Sophora japonica L Rutin has been proven having many

biological effects as inflammatory, antioxidant, etc [29]

Compound 13, an yellow powder, showed the pseudo molecular ion peak at m/z = 623

[M-H]- in the negative ESI-MS Its molecular formula was deduced as C28H32O16 (M = 624) from

MS and NMR data The NMR data of 13 were very similar to those of 12 with only one difference The signal of hydroxy in ring B of 12 was replaced by methoxy signals (δH = 3.96 s,

3H and δC = 56.8) in compound 13 together with the downshifted of C-3’ (∆δC = 2.67) when

compared to those of compound 12 Finally, the structure of 13 was established as

isorhamnetin-3-O-β-rutinoside by comparison with the published spectroscopic data in [30] The other name

of this compound is narcissin

Table 2 1H and 13C-NMR (500 and 125 MHz) of compounds 7 – 10 (CD3OD).

7

δH (J, Hz)

8

δH (J, Hz)

9

δH (J, Hz)

10

δH (J, Hz)

7

δC

8

δC

9

δC

10

δC

1 5.97 d, 1.0 5.97 d, 1.5 5.83 br s 5.07 d, 9.0 93.3 CH 93.3 98.3 101.2

6.5

5.58 dd,

1.5 & 6.5

10 4.69 dd,

14.0 & 1.0

4,80 dd,

14.0 & 1.0

4.21 br s 4.21 br d,

15.0

4.36 br d,

15.0

4.97 br d,

14.5 4.83

br d, 14.5

61.9 CH2 60.1 61.0 63.8

Glc-2 3.22 dd,

9.0 & 8.0 3.21 dd, 9.0 & 8.0

3.41 br t,

8.5

m 3.30 – 3.60 m 3.20 – 3.34 m

3.25 – 3.33

m

Glc-6 3.94 dd,

12.0 & 2.0

3.70 dd,

12.0 & 6.0

3.94 dd,

12.0 & 2.0

3.84 dd,

12.0 & 6.0

3.88 br d,

11.5

3.66 br d,

11.5

3.87 br d,

10.0 3.64

dd, 5.0, 12.0

4 CONCLUSION

In summary, this report deals with the isolation and structural elucidation of thirteen

compounds from the n-hexane, ethyl acetate and n-butanol extracts of Hedyotis pinifolia Three

of them (compounds 1, 3 and 11) were obtained for the first time from this genus